Abstract
Since the first descriptions of electrophoresis in small diameter tubes in the 1970s and 1980s (1,2), capillary electrophoresis (CE) has been recognized for its potential to replace slab-gel electrophoresis for the analysis of nucleic acids (3,4). In particular, the availability of commercial instrumentation for CE over the last several years has made both the size determination and quantitation of DNA restriction fragments or polymerase chain reaction (PCR) products amenable to automation. Due to the same charge-to-mass ratio, the electrophoretic mobility of nucleic acid molecules in free solution is largely independent of their molecular size (5). Therefore, a sieving medium is required for the electrophoretic analysis of DNA fragments based on their size. Typically, two different principal types of separation matrix are used. The first type of matrix is of high viscosity polymer (e.g., polyacrylamide) with a well-defined crosslinked gel in regard to the structure and size of its pores. The second type of matrix is a noncrosslinked linear polymer network of materials such as, linear polyacrylamide, agarose, cellulose, dextran, poly(ethylene oxide), with lower viscosity than the former type and with a more dynamic pore structure. Although the first type of matrix is attached covalently to the capillary wall and may provide better separation for small (sequencing) fragments, the second matrix format has the advantage of being able to be replenished after each electrophoretic cycle. This typically extends the lifetime of a capillary, prevents contamination of the system, avoids sample carryover and allows the use of temperatures well above room temperature. Most matrices used in both systems are tolerant to the addition of DNA denaturants. Many different media useful for the separation of DNA have now become commercially available (6).
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Mikkers, F. E. P., Everaerts, F. M., and Verheggen, T. P. E. M. (1979) High performance zone electrophoresis. J. Chromatogr. 169, 11–20.
Jorgenson, J. W. and Lukacs,l K. D. (1981) Zone electrophoresis in open tubular glass capillaries. Anal. Chem. 53, 1298–1302.
Landers, P. J., Oda, R. P., Spelsberg, T. C., Nolan, J. A., and Ulfelder, K. J. (1993) Capillary Electrophoresis: A powerful microanalytical technique for biologically active molecules. BioTechniques 14, 98–111.
Karger, B. L., Chu, Y. H., and Foret, F. (1995) Capillary electrophoresis of proteins and nucleic acids. Annu. Rev. Biophys. Biomol. Struct. 24, 579–610.
Olivera, B. M., Baine, P., and Davidson, N. (1964) Electrophoresis of nucleic acids. Biopolymers 2, 245–257.
Wehr, T., Zhu, M., and Mao, D. T. (2001) Sieving matrix selection, in Capillary Electrophoresis of Nucleic Acids, Vol. 1 (Mitchelson, K. R., and Cheng, J., eds.), Humana Press, Totowa, NJ, pp. 167–187.
Mueller, O., Minarik, M., and Foret, F. (1998) Ultrafast DNA analysis by capillary electrophoresis. Electrophoresis 19, 1436–1441.
Muth, J., Williams, P. M., Williams, S. J., Brown, M. D., Wallace, D. C., and Karger, B. L. (1996) Fast capillary electrophoresis-laser induced fluorescence analysis of ligase chain reaction products: Human mitochondrial DNA point mutations causing Leber’s hereditary optic neuropathy. Electrophoresis 17, 1875–1883.
KlĂ©parnĂk, K., MalĂ , Z., HavĂ ÄŤ, Z., Blazkova, M., Holla, L., and BoÄŤek, P. (1998) Fast detection of a (CA)18 microsatellite repeat in the IgE receptor gene by capillary electrophoresis with laser-induced fluorescence detection. Electrophoresis 19, 249–255.
Atha, D. A., Wenz, H.-M., Morehead, H., Tian, J., and O’Connell, C. (1998) Detection of p53 point mutations by single strand conformation polymorphism (SSCP): analysis by capillary electrophoresis. Electrophoresis 19, 172–179.
Brinson, E. C., Adriano, T., Bloch, W., Brown, C. L., Chang, C. C., Chen, J., et al. (1997) Introduction to PCR/OLA/SCS, a multiplex DNA test, and its application to cystic fibrosis. Genetic Testing 1, 61–68.
Orita, M., Iwahana, H., Kanazawa, H., Hayashi, K., and Sekiya, T. (1989) Detection of polymorphisms of human DNA by gel electrophoresis as a single-strand conformation polymorphism. Proc. Natl. Acad. Sci. USA 86, 2766–2770.
Wenz, M.-H., Robertson, J. R., Menchen, S., Oaks, F., Demorest, D. M., Scheibler D., et al. (1998) High Precision Genotyping by Denaturing Capillary Electrophoresis. Genome Res. 8, 69–80.
Landegren, U., Nilsson, M., and Kwok, P. Y. (1998) Reading bits of genetic information: methods for single-nucleotide polymorphism analysis. Genome Res. 8, 769–776.
Syvänen, A.-C., Aalto-Setala, K., Harju, L., Kontula, K., and Soderlund, H. (1990) A primer-guided nucleotide incorporation assay in the genotyping of apolipoprotein E. Genomics 8, 684–692.
Landegren, U., Kaiser, R., Sanders, J., and Hood, L. (1988) A ligase-mediated gene detection technique. Science 241, 1077–1080.
Grossman, P. D. Bloch, W., Brinson, E., Chang, C. C., Eggerding, F. A., Fung, S., et al. (1994) High-density multiplex detection of nucleic acid sequences: oligonucleotide assay and sequence-coded separation. Nucleic Acids Res. 22, 4527–4534.
Day, D. J. Speiser, P. W., White, P. C., and Barany, F. (1995) Detection of steroid 21-hydroxylase alleles using gene-specific PCR and a multiplexed ligation detection reaction. Genomics 29, 152–162.
Hayashi, K., Wenz, H.-M., Inazuka, M., Tahira, T., Sasaki, T., and Atha, D. H. (2001) SSCP analysis of point mutations by multicolor capillary electrophoresis, in Capillary Electrophoresis of Nucleic Acids, Vol. 2 (Mitchelson, K. R., and Cheng, J., eds.), Humana Press, Totowa, NJ, pp. 109–126.
Ren, J. (2001) SSCP analysis by capillary electrophoresis with laser-induced fluorescence detector, in Capillary Electrophoresis of Nucleic Acids, Vol. 2 (Mitchelson, K. R., and Cheng, J., eds.), Humana Press, Totowa, NJ, pp. 127–134.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2001 Humana Press Inc.
About this protocol
Cite this protocol
Wenz, H.M., Dailey, D., Johnson, M.D. (2001). Development of a High-Throughput Capillary Electrophoresis Protocol for DNA Fragment Analysis. In: Mitchelson, K.R., Cheng, J. (eds) Capillary Electrophoresis of Nucleic Acids. Methods in Molecular Biology™, vol 163. Humana Press. https://doi.org/10.1385/1-59259-116-7:3
Download citation
DOI: https://doi.org/10.1385/1-59259-116-7:3
Publisher Name: Humana Press
Print ISBN: 978-0-89603-765-6
Online ISBN: 978-1-59259-116-9
eBook Packages: Springer Protocols